The major objectives of this proposal are the isolation and characterization of a comprehensive series of deletion mutants of human adenoviruses 2 and 5, and their use to probe the process of regulation of viral gene expression, viral transformation, and viral DNA replication. Deletion mutants obtained from stocks containing naturally arising defective adenovirus variants will be cloned using defective helper viruses, a series of complementing cell lines constructed for the purpose, and each mutant will be characterized biochemically. Mutants unable to transform cells, mutants which appear to have aberrant regulatory phenotypes, and mutants impaired in viral DNA replication will be examined in detail to define the viral genes and sites important in these processes. In a related series of experiments, site-specific mutagenesis will be employed to generate point mutations in the terminal 50-100 bases of the adenoviral genome, the region which seems to contain the origins of viral DNA replication. The detailed characterization of such mutants should make it possible to confirm this suggestion, and to construct a high-resolution functional map of the terminus of the viral DNA molecule.